CN111544584A - Therapeutic effect of a monoclonal antibody on Parkinson's disease - Google Patents

Abstract

The invention relates to the therapeutic effect of a monoclonal antibody on Parkinson's disease. The monoclonal antibody is an anti-serine 129-position phosphorylated alpha synuclein (p-α-synuclein, p-α-syn) monoclonal antibody ( C140S), the present invention particularly relates to the research on the effect of this antibody in the treatment of abnormal activation of immune cells.

Description

Therapeutic effect of a monoclonal antibody on Parkinson's disease

technical field

The invention relates to the therapeutic effect of a monoclonal antibody on Parkinson's disease. The monoclonal antibody is an anti-serine 129 phosphorylated alpha synuclein (p-α-synuclein, p-α-syn) monoclonal antibody ( C140S), the present invention particularly relates to the research on the effect of this antibody in the treatment of abnormal activation of immune cells.

Background technique

Parkinson's Disease (PD) is a common neurodegenerative disease characterized by motor symptoms such as bradykinesia, muscle rigidity, resting tremor, and postural instability, as well as other neuromodulatory and neurotransmitter systems. Also affected, resulting in a variety of non-motor symptoms such as cognitive decline, depression, sleep disturbance, smell disturbance, urination and gastrointestinal abnormalities; the pathogenesis of Parkinson's disease is not fully understood, the main pathological features It is the progressive death of dopaminergic neurons in the substantia nigra of the midbrain, and the aggregation of α-synuclein (α-syn) in neurons to form Lewy bodies, the main component of which is the phosphorylation of alpha synucleon at serine 129. protein (p-α-syn).

The current treatment method for PD is mainly drug treatment. The most classic drug in drug treatment is levodopa, but levodopa can only improve motor symptoms. With the prolongation of use time and the increase of dosage, the toxic and side effects of these drugs become more and more The bigger the disease, the more curative effect and “on-off” phenomenon appear in most PD patients. Another class of drugs are enzyme inhibitors such as MAO-B inhibitors, which can reduce dopamine metabolism by specifically inhibiting monoamine oxidase B, but Some patients experience side effects such as involuntary movements. Deep brain stimulation (DBS) therapy is aimed at patients with tremor symptoms, using a certain frequency of electrical signals to stimulate the subthalamic nucleus, so as to relieve movement disorders. These treatments are all aimed at the treatment of motor symptoms, cannot really delay the progression of the disease, and have certain limitations on the indications of the disease, so immunotherapy with strong etiology specificity shows its advantages. Antibodies can be used as drugs to target and recognize the target protein that causes the disease, promote the clearance of the target protein, reduce the aggregation of the target protein and prevent the spread of the target protein to play a therapeutic role.

In Parkinson's disease, the target protein for immunotherapy treatment is alpha-synuclein. Abnormal aggregated forms of α-synuclein, as well as phosphorylation modifications, can cause cytotoxicity and spread from cell to cell, causing cell death and Parkinson's disease. At present, the antibodies against α-syn include antibodies against α-syn in different aggregation states (anti-monomer, anti-oligomer, anti-fibrotic antibodies); α-syn is divided into three domains, N-terminal, NAC region and C-terminus, so there are also antibodies against different regions of α-syn (anti-N-terminus, anti-C-terminus, anti-NAC region antibodies), but none of them have been used clinically. The toxic effect of α-synuclein is mainly related to its phosphorylation modification. The alpha-synuclein phosphorylated at the 129th position of serine is more damaged to cells, and it is easier to aggregate and spread, making it more difficult to be degraded. , causing cytotoxic and neurodegenerative changes. The use of monoclonal antibodies specific for phosphorylated alpha-synuclein at position 129 of serine to neutralize toxic proteins, thereby reducing their cytotoxicity, is a novel etiology-specific therapeutic approach.

Anti-serine 129 phosphorylated alpha synuclein (p-α-synuclein, p-α-syn) monoclonal antibody (abbreviation: C140S): disclosed in Chinese patent ZL201610913085.1, the patent specification describes the antibody Source: In order to establish the ELISA detection method of the present invention, a mouse-derived monoclonal antibody C140S against human α-syn phosphorylated at position 129 was prepared. The preparation method of the monoclonal antibody is as follows: use P1 (M18631-1hz-1-1, Ac-CEAYEMP(pS)EGG-NH2, 123-131 peptide fragment of human-derived serine phosphorylated α-syn at position 129) peptide fragment immunized balb-c mice, spleen was taken to prepare fusion bone marrow Tumor cell line, a cell line with clone number C140S was screened by indirect ELISA, and the cell line was injected into the abdominal cavity of 4 balb-c/nu mice. After ELISA verifies the reactivity of the purified antibody (i.e. 0.125ug/ml antibody recognizes 1000ng/ml positive group absorbance at 490nm wavelength is at least 30 times higher than negative group, and the background and negative group readings are less than 0.2), can be used As the detection antibody in this method, the final concentration is 4 mg/ml.

(This cell line has been preserved in the China General Microorganism Culture Collection and Management Center, numbered CGMCC12993, dated September 26, 2016, classified name: Antibody Hybridoma, Address No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing ).

SUMMARY OF THE INVENTION

The invention provides the application of an anti-serine 129 phosphorylated alpha synuclein monoclonal antibody (C140S) in the preparation of a medicine for treating Parkinson's disease, wherein the application is based on that the antibody can block p-α-syn in cell-to-cell transmission.

The present invention further provides a pharmaceutical preparation, which comprises an anti-serine 129 phosphorylated alpha synuclein monoclonal antibody (C140S), wherein the monoclonal antibody is of the type: IgG, and the antibody subtype is: IgG2b . The pharmaceutical formulations can be administered to mammals, including humans, in any manner. Parenteral administration is preferred, with injection being particularly preferred.

The application of the present invention is that the antibody can block the propagation of p-α-syn between cells.

The application of the present invention means that the antibody can pass through the blood-brain barrier and enter the brain parenchyma within 6 hours.

The application of the present invention is that the antibody can improve the inversion of the ratio of CD4+/CD8+ T cells, relieve the behavioral disorder of the transgenic mice, increase the rod retention time of the transgenic mice, increase the limb strength of the mice, and enhance the Coordination in transgenic mice.

The application of the present invention is that the antibody can neutralize the toxic protein and reduce its cytotoxicity.

The application of the present invention is that the antibody can reduce the content of p-α-syn in the brain of transgenic animals, increase the content of TH in the brain, and alleviate the avoidance obstacle of the transgenic mice, indicating that the antibody C140S has a neuroprotective effect, and It has a therapeutic effect on PD models.

In order to prove the purpose of the present invention, the present invention uses the anti-serine 129 phosphorylated alpha synuclein monoclonal antibody (C140S) as the experimental drug, and studies the relevant Parkinson's disease model, and the research results are as follows:

The cell model used was HEK293T cell line overexpressing α-syn.

Specific steps are as follows:

1) The α-syn gene was transfected into HEK293T cells, and the cell supernatant was collected after 24 hours for indirect ELISA experiment. The results showed that there was secreted p-α-syn in the cell supernatant.

2) The aggregation of the N-terminus and C-terminus of Luciferase will cause coelenterazine to emit fluorescence. The N- and C-segments of Luciferase were connected to α-syn and then transfected into HEK293T cells respectively, and the transfected α was collected after 24 hours. The supernatant of -syn-luciferase-N medium was added to the culture medium of cells transfected with α-syn-luciferase-C. After 24 hours, coelenterazine was used for color development. The results showed that p-α-syn could spread between cells.

3) After adding C140S to the supernatant of α-syn-luciferase-N medium, the intracellular fluorescence intensity of the transfected α-syn-luciferase-C plasmid decreased significantly, indicating that C140S played a role in the propagation of p-α-syn. blocking effect.

The present invention further provides a method for treating PD transgenic animal model with anti-serine 129 phosphorylated alpha synuclein monoclonal antibody (C140S).

The PD animal model used in the present invention is a transgenic animal overexpressing α-syn.

Specific steps are as follows:

1) After intraperitoneal injection of rhodamine-labeled C140S in wild-type mice, in vivo imaging was used to detect the fluorescence intensity in the mouse brain. The results showed that the antibody drug C140S labeled with rhodamine can enter the brain through the blood-brain barrier within 6 hours. in substance.

2) One week after transgenic mice were intraperitoneally injected with 10 mg/kg antibody C140S, the ratio of CD4 ⁺ /CD8 ⁺ T cells in the plasma of each group of mice was detected by flow cytometry. The results proved that antibody drug C140S can improve CD4 ⁺ /CD8 ⁺ The inversion of the proportion of T cells can alleviate the behavioral disorder of the transgenic mice, increase the rod retention time of the transgenic mice, increase the strength of the limbs of the mice, and enhance the coordination ability of the transgenic mice.

3) Stereotaxic injection of C140S antibody into the dorsal striatum of the transgenic mouse brain was performed to detect the contents of p-α-syn and tyrosine hydroxylase (TH) in the mouse brain and the motor behavior of the mouse. The results show that the antibody drug C140S can reduce the content of p-α-syn in the brain of transgenic animals, increase the content of TH in the brain, and relieve the avoidance barrier of the transgenic mice. effect.

The advantages of the invention are that after intraperitoneal injection of C140S antibody drugs in PD animal models, the changes in abnormal immunity in peripheral blood can be alleviated. Motor behavior disorders in mice.

Description of drawings:

Figure 1 P-α-syn can spread between cells, and the process of spreading can be blocked by the antibody drug C140S. HEK293T cells were transfected with α-syn plasmid, and the cell supernatant was collected for 24 hours, concentrated 10 times, and then tested by ELISA. The results showed that the α-syn in the supernatant of the overexpression α-syn group was significantly more than that of the control group; B. The luciferase was removed Divided into N-terminal and C-terminal, respectively connected with α-syn and then transfected into HEK293T cells by plasmid, collected the supernatant of α-syn+luciferase-N-terminal group, concentrated and added to α-syn+luciferase-C-terminal group, After 24 hours, coelenterazine was added, and the autofluorescence intensity was detected under a microplate reader; C. Collect the transfected α-syn+luciferase-N-terminal cell protein, and use the WB experiment to detect the expression of α-syn protein and phosphorylated α- syn protein (17kd), also expresses α-syn ⁺ luciferase-N and phosphorylated α-syn+luciferase-N protein (27kd); D. Using a microplate reader to detect autofluorescence, it was found that transfected α-syn+luciferase -Add 50μl of concentrated α-syn+luciferase-N-terminal group cell supernatant to the cells of the C-terminal group. After the addition of coelenterazine, the fluorescence intensity increased significantly. Adding phosphorylated α-syn antibody c140s to the concentrated supernatant can neutralize the spread During the process, the fluorescence intensity of p-α-syn+luciferase-N decreased significantly, and the difference between adding other volumes of concentrated supernatant and antibody was not obvious; E. Simultaneous transfection of α-syn+luciferase-C and α-syn+luciferase into HEK293T cells -N, the fluorescence intensity increased significantly after color development with coelenterazine. At this time, the addition of C140S antibody to the cell supernatant did not affect the luminescence of luciferase; F. Due to the lack of aggregation properties of α-syn, only transfected luciferase-N-terminal and The C-terminus cannot fluoresce.

Figure 2: The half-life of antibody C140S in mice is 5 days, and intraperitoneal injection can reduce the content of p-α-syn in the peripheral blood of transgenic mice. A. Inject 10 mg/kg of p-α-syn antibody drug (C140S) into the abdominal cavity of TG mice, and take the peripheral blood on the 1st, 3rd, 5th, 7th, and 9th days to detect C140S and p- The amount of α-syn; (B, C) The amount of p-α-syn in peripheral blood decreased significantly after the 9th day after administration.

Figure 3: Intraperitoneal injection of antibody C140S alleviates abnormal immune responses in transgenic mice.

A: The ratio of CD4 ⁺ /CD8 ⁺ cells in the peripheral plasma of transgenic animals was lower than that of wild-type mice; B, C: One week after intraperitoneal injection (INTRA) of 10 mg/kg antibody C140S, the number of CD8 ⁺ T cells in peripheral plasma was significantly reduced, and the number of CD4 + T cells in peripheral plasma was significantly reduced. The ⁺ /CD8 ⁺ ratio was significantly increased.

Figure 4: Intraperitoneal injection of antibody C140S can cross the blood-brain barrier in transgenic mice. After conjugating rhodamine as a fluorescent label with C140S antibody and intraperitoneal injection, A: the degree of C140S diffusion in mice was detected in vivo; B: 4h, 5h, and 6h of the mice were taken for imaging after cerebral hemorrhage, and it was found that at 4h Rhodamine-labeled C140S has spread to the brain parenchyma, and the amount of the drug entering the brain gradually increases over time.

Figure 5: P-α-syn in the peripheral blood and brain parenchyma of transgenic mice was reduced after brain-localized injection of C140S, and the loss of TH-positive neurons in the mouse brain was reduced. A: The number of TH-positive neurons on the C140S drug-injected side was significantly more than that on the control side; B: Stereotactic injection of commercially available α-syn antibody as a control antibody, C140S as the experimental group was treated with antibody drugs, and then the striatal soluble protein was extracted for Western blotting Experiment; C, D: The results showed that the expression of TH in the TG group was significantly lower than that in the WT group. The expression of TH protein in the C140S antibody drug group was significantly higher than that in the TG group, while the expression of TH in the control α-syn antibody group was similar to that in the TG group. There is no significant difference compared to.

Figure 6: Significant reduction of p-α-syn in the striatum of transgenic mice following brain-localized injection of C140S. A: The soluble and insoluble protein fractions were extracted respectively and subjected to Western blot experiment; B, C: The content of p-α-syn in the soluble and insoluble protein fractions of the TG group after C140S treatment was significantly lower than that of the TG group, while the -syn antibody injection did not reduce p-α-syn content.

Figure 7: C140S treatment can alleviate dyskinesia in transgenic mice

A. Use 10-12 month old mice to perform the rotarod fatigue test, do accelerated exercise at a speed of 10-60r/min, record the exercise time and find that the TG mice rotarod time is 136±4.427 seconds, which is 166.2 seconds with the WT group Compared with ±6.339 seconds, it was significantly decreased, and the animal rotarod time after intraperitoneal injection was 152.1 ± 5.123 seconds, which was significantly different from the TG group, indicating that there was a therapeutic effect; B. Use 10-12 month old mice to conduct open field experiments , normal mice are afraid of open spaces and are more willing to walk against the wall. After statistics, it was found that the ratio of the time spent in the surrounding area in the TG group to the total activity time was about 0.81±0.01, which was significantly lower than that of the WT group, which was 0.98±0.02, indicating that its The avoidance was changed. After the stereotaxic injection of antibody treatment, the avoidance was improved, and the time ratio was 0.96±0.03, which was significantly improved compared with the TG group. Strength, according to the scoring table, the success of drilling up the net +3 points, the four limbs grasping the net for 10 seconds +2 points, the double forelimbs grasping the net for 10 seconds +1 point, falling within 10 seconds is 0 points, after statistics, it is found that the TG group scores 2.333±0.4216 points, significantly lower than WT group 5±0.4472 points, and 2.833±0.4773 points of intraperitoneal administration group, no difference compared with TG group, indicating that limb strength did not recover; D. Using 10-12 months old The rod climbing test was performed to evaluate the coordination ability of the mice. Statistics showed that the TG group took 16.23 ± 2.684 seconds to climb from the top to the bottom, which was significantly higher than that of the WT group, 8.39 ± 1.438 seconds. The intraperitoneal administration treatment group took 11.62 ± 1.596 seconds and There was no difference compared to the TG group.

Detailed ways

The present invention will be further described below in conjunction with specific implementation examples, but the present invention is not limited to the following examples. The cell lines used in the following examples are as follows:

Thy-1-α-syn transgenic mice: purchased from Jackson Laboratory, USA, animal license number: 3377883HEK293T cell line: human embryonic kidney cells (frozen in our laboratory)

Example 1:

1. Indirect ELISA to compare p-α-syn content in cultured cell supernatant

(1) Preparation of related reagents

A. Coating solution: CBS pH 9.6 500ml (Na2CO3 0.84g, NaHCO3 1.45g, H2O 500ml) stored at 4°C.

B. Washing solution and dilution solution (mother solution): PBST pH 7.4 1000ml (10×) (NaH2PO4·2H2O 2.964g, Na2HPO4·12H2O 28.998g, NaCl2 9.000g, H2O 1000ml).

C. 20% Tween-20 100ml (Tween-20 20ml, H2O 80ml), autoclaved and stored at room temperature.

D. Washing solution and diluting solution (working solution): 50ml PBST pH 7.4, 1ml Tween-20 to make up to 500ml.

E. Blocking solution: 5% BSA, 25ml (BSA1.25g, diluent 25ml, ready for use)

F. Substrate solution: OPD (o-phenylenediamine)-H2O2 preparation, phosphate-citric acid buffer 0.1M, respectively take 4.86ml and mix 5.14ml

G. Color developer (currently used and prepared): take out 10ml F solution+4mg OPD+5μl H ₂ O ₂

H. Stop solution: concentrated H ₂ SO ₄ 20ml+H ₂ O 80ml

(2) Experimental steps:

A. ELISA plate coating: Dilute the antigen with coating solution, add 100 μl per well to a 96-well plate, overnight at 4°C;

B. Wash the plate with 200μl washing solution for 5 minutes, wash 3 times, and pat dry;

C. Seal the plate with 200 μl/well of blocking solution, block at 37°C for 2 hours, and wash the plate 3 times;

D. Add 100 μl of C140S antibody diluted with diluent;

E. After incubating at 37°C for 2 hours, wash the plate 3 times;

F. Add 100 μl HRP-labeled anti-mouse IgG, incubate at 37°C for 1 hour, wash the plate 3 times,

G. Add substrate color reagent, develop color at 37°C for 30 minutes; add stop solution;

H. Use a microplate reader (Victor3) to measure the absorbance value at 490nm wavelength.

2: α-syn-luciferase-C and α-syn-luciferase-N plasmid extraction

(1) Reagent preparation:

LB liquid medium configuration

tryptone 10g Bacterial Yeast Extract 5g NaCl 10g Deionized water 950ml

Adjust the pH value to 7.0 with 5M NaOH, then dilute to 1L with deionized water, sterilize by autoclaving at 122°C for 20min, cool down for use, and add ampicillin (final concentration: 100μg/ml) or cardboard on a sterile operating table. Namycin (final concentration of 50 μg/ml) was mixed, sealed and stored at room temperature. Configuration and plate preparation of solid LB medium

tryptone 2g Bacterial Yeast Extract 1g NaCl 2g agar for bacterial culture 3g

Deionized water to 200ml, autoclaved at 122°C for 20min, cooled to 50°C, added ampicillin (final concentration of 100μg/ml) in a sterile operating table, mixed well, poured 5ml into a sterile glass petri dish, and dried. Store at 4°C after drying.

(2) The plasmid was transformed into highly competent cells.

A. Gently add 5 μl of the ligation reaction to DH5α-E.coli (100 μl, taken out of the -80°C refrigerator before use, thawed on ice), and mixed gently. Incubate on ice for 30min;

B. Put it in a water bath at 42°C for 60-90s, and quickly put it on ice for 2min.

C. Add 400 μl of ampicillin-free LB liquid medium preheated to room temperature, put it into a horizontal shaker, shake at 200 rpm, and cultivate at 37 °C for 40 min;

D. After shaking, take 200 ml of bacterial liquid from the reaction tube, spread it on an ampicillin-resistant LB culture plate preheated at room temperature, and cultivate at 37°C for 12-16 hours.

(3) Extract plasmid with Wizard R Plus Midipreps DNAPurification System kit

A. Scratch the bacterial solution reserved for the correctly sequenced group on a petri dish, incubate at 37°C for 12 hours, and randomly pick a single clone colony (separately and not cross with other colonies) and inoculate it in advance by adding 3ml of ampicillin or cardboard. In the LB medium of namycin, ensure good ventilation, shake at 37°C, 250rpm for 12h.

B. Take 1ml of the bacterial solution in the shaking tube and add it to 100ml of LB medium containing ampicillin or kanamycin, at 37°C, 250rpm, shake for 12-16h, and mix 1ml of bacterial solution with glycerol in each group (according to the ratio of 4:1 Mixed) to keep the strains, and the rest of the strains were used for plasmid extraction.

C. Pour the bacterial liquid into a 50ml large centrifuge tube, centrifuge at 10000g at 4°C for 10 minutes to enrich the bacteria, discard the supernatant, invert the centrifuge tube on absorbent paper, and repeat twice to collect the bacteria.

D. Add 3ml of Cell Resuspnsion Solution, completely resuspend the bacterial solution, then add 3ml of LysisSolution, invert and mix 4 times until the bacterial solution is clear. Add 3ml of neutralization, invert upside down and mix 4 times, let stand for 5min, 4 ℃, 14,000g centrifugation for 15min, if the precipitate is not firmly attached, centrifuge again under the same conditions, transfer the supernatant to another sterile centrifuge tube (avoid Aspirated white precipitate). Add 10ml of complete mixing resin (DNA Purification Resin), invert upside down 3 times, let stand for 2min, repeat 2-3 times. Transfer the above mixture into midicolumn, suction with vacuum negative pressure, pour 15ml first and then add continuously until the mixture passes through, add 15ml Column Washing Solution, and wash again. Cut the midicolumn into a sterile EP tube and centrifuge at 12,000g for 2 min to remove the residual Washing Solution in the midicolumn.

Put the midicolumn in another sterile EP tube, add 150 μl of preheated sterile deionized water at 60°C, incubate at 60°C for 2 minutes, and centrifuge at 12,000g for 2 minutes to collect the first batch of plasmids. Then put the midicolumn in another sterile EP tube, add 150 μl of preheated sterile deionized water, incubate at 60 °C for 2-3 min, centrifuge at 12,000 g for 2 min, collect the second batch of plasmids, and put the collected samples in each tube. Centrifuge at 12,000g for 5min. E. The plasmid concentration was detected by Nanodrop, and the obtained plasmid was stored at -20°C for cell transfection.

3. HEK-293T cells were transfected with α-syn-luciferase-C and α-syn-luciferase-N plasmids

A. Seed 1x10 ⁶ cells in a 90nm dish,

B. Add 375 μl of OPTI-MEM into the transfection tube, add 20 μl of the transfection reagent LipofectamineTM2000, shake while adding, and let stand at room temperature for 5 minutes. 375 μl of OPTI-MEM was added to the transfection tube, 12 μg of the target plasmid was added into it, shaken while adding, and left at room temperature for 5 min.

C. The two liquids were mixed and shaken gently, and allowed to stand at room temperature for 20 min.

D. Aspirate the intracellular medium, wash the cells three times with OPTI-MEM to remove serum, and then add OPTI-MEM, 7.5 ml per culture dish.

E. Add the mixture of plasmids and transfection reagents to the petri dish, incubate in a 37°C, 5% CO ² incubator for 6 hours, replace with normal medium, and then process the cells for the corresponding time.

4. P-α-syn propagation and blocking experiments

(1) Experimental reagents: Luciferase N-terminal plasmid and Luciferase C-terminal plasmid (constructed in our laboratory), Coelenterazine (BD Company)

(2) Luciferase is a protein that produces autofluorescence under the stimulation of coelenterazine. The N-terminus and C-terminus of Luciferase were respectively linked to α-syn to construct a plasmid, which was transfected into HEK293T cells.

(3) After 24 hours, collect the supernatant of cells transfected with Luciferase N-terminal-α-syn, use a concentration tube to concentrate 10 times, and then add it to the culture dish of cells transfected with Luciferase C-α-syn. C140S antibody as blocking drug.

(4) Coelenterazine was added after 24 hours, and the intensity of autofluorescence was observed in a microplate reader.

Example 2

1. Detection of CD4 ⁺ , CD8 ⁺ T cells in plasma of transgenic mice

(1) Take blood from the inner canthus of mice, add 3 times the volume of red blood cell lysate, centrifuge at 2,000g for 15 minutes, discard the supernatant, add 2 times the volume of red blood cell lysate, resuspend and centrifuge again at 2,000g for 15 minutes, discard the supernatant to retain the pellet .

(2) Live dead staining, add 0.1 μL of Zombie Green TM Dye to each tube, shake and mix, incubate in the dark at room temperature for 15 min, wash with Wash Buffer, centrifuge at 1 500g for 5 min, and discard the supernatant.

(3) Blocking of Fc receptors: 1 μg/10 ⁶ cells of Anti-Mouse CD16/CD32 antibody was added to each tube to block non-specific staining caused by fluorescent antibody Fc receptors. Incubate with shaking at 4°C for 30 min.

(4) Add the corresponding surface staining antibody to each tube, and incubate at 4°C for 30 minutes in the dark with shaking.

(5) Add 100 μL of fixative for 30 min.

(6) Add 200 μl of permeabilization solution to wash the cells, and centrifuge at 600 g for 5 min at room temperature.

(7) 100 μl of permeabilization solution was added to resuspend the cells, and 1 μl of antibody was added to stain intracellular proteins for 30 min at room temperature.

(8) Add 200 μl of permeabilization solution, centrifuge at 600 g for 5 min at room temperature, and discard the supernatant.

(9) Wash once with PBS, add 1 ml of PBS to resuspend the pellet, and test on the machine.

2. Small animal in vivo imaging experiments

(1) Experimental equipment: Kodak small animal imager, depilatory cream (Rou Ting).

(2) The excitation light of rhodamine is 590 nm, and rhodamine is connected to C140S.

(3) C140S labeled with rhodamine was injected into mice by intraperitoneal injection, and then the mice were anesthetized with 1.5 ml of 10% chloral hydrate, and the chest and abdomen were depilated to avoid affecting imaging, and then the mice were fixed on a photographic plate.

(4) Use the X-ray function of the in vivo imager to first save the outline of the mouse, then adjust to the appropriate excitation light and emission light, and scan every 1 hour.

(5) In order to avoid the shielding effect of the skull, after 4h, 5h, 6h, and 7h of administration, the mice in each group were perfused with normal saline, and the brain tissue was taken out for imaging to observe the drug entering the brain parenchyma through the blood-brain barrier. 3: Detection of C140S concentration in the plasma of transgenic mice:

A. Plate wrapping: take avidin and dilute it with CBS, add each 100 μl to a 96-well plate, overnight at 4°C;

B. Wash the plate with 200μl washing solution for 5 minutes, wash 3 times, and pat dry;

C. 100 μl of C140S serum protein with 100 μl biotin label diluted with diluent;

D. After incubation at 37°C for 2 hours, wash the plate 3 times;

E. Add alkaline phosphatase-labeled anti-mouse IgG diluted in solution D at 1:10000, 100 μl per well, and incubate at 37°C for 1 hour in the dark;

F. Washing 2 times;

G. Add 100 μl of pNPP to each well and let stand at 37°C for 1 hour for color development;

H. On a PerkinElmer Viktor 3 microplate reader, set to read the absorbance value at 405nm, and illuminate each well for 0.1 seconds.

I. Measure the absorbance value at 490nm wavelength with a microplate reader (Victor3).

4: Extraction of protein components from transgenic mice

(1) Cut 40 mg of fresh brain tissue into a 1.5 ml EP tube, add 200 μl of RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors), and operate on ice throughout.

(2) Grind the tissue into a tissue homogenate using a homogenizer, and then sonicate the tissue under a sonicator.

(3) Lysis on ice for 30min.

(4) The lysate was centrifuged at 4° C. centrifuge at a speed of 12,000 g for 30 min, and the supernatant was collected as a soluble protein fraction, and the precipitate was an insoluble protein fraction.

(5) After measuring the protein concentration in the supernatant, add loading buffer, denature at 95°C for 10min, and store at -20°C.

(6) 10M urea was added to the precipitation part, and after mixing, it was crushed under an ultrasonic instrument.

(7) After dissolving the insoluble protein, add it directly to the loading buffer, denature it at 95°C for 10 minutes, and store it at -20°C. 5: Transgenic mice were stereotaxically injected with antibody C140S and commercially available α-syn antibody.

(1) 10-month-old transgenic mice were intraperitoneally injected with 0.1 ml of 10% chloral hydrate to ensure that they were under shallow anesthesia, which was helpful to improve the postoperative survival rate.

(2) Fix the mouse on the stereotaxic apparatus, and remove the hair on the top of the mouse head with a blade.

(3) Make a longitudinal incision on the top of the mouse head with a scalpel, expose the bregma, adjust the position of the microinjector, the bregma + 0.2mm, the midline + 2.0mm, and the positioning mark is made with an oil pen.

(4) Clean the surgical instruments with medical alcohol and normal saline, and drill holes on the marked sites with a dental drill.

(5) Lower the microinjector to the position of the hole, adjust the zero and then insert the needle 2.6mm, the position is the right striatum. (6) Inject 1 μl of antibody at a rate of 0.1 μl per minute, and retain the needle for 10 minutes after injection.

(7) After pulling out the needle, clean the wound and suture, pay attention to keeping the mice warm, and observe the vital signs.

6: Animal behavior testing

(1) Rotor fatigue test

In order to keep the body balance on the rotating rod and not fall, the mouse needs to move in the opposite direction of the rotating rod, and maintain the coordinated movement of the limbs muscles, otherwise it will fall off the rod, and the mice with dyskinesia or poor coordination will be more likely to suffer. Fall down quickly. By statistical analysis of the quantitative index of the drop time of the mice, the movement disorder or the relief of the disorder in the mice can be evaluated. In order to evaluate the endurance of the mice, the initial speed of the rotarod fatigue instrument was set to 10 rpm/min, the acceleration was 9.0, and the maximum speed was 60 rpm/min. .

(2) Drilling mesh grip strength test

In order to evaluate the grip strength and coordination ability of the mice, the diameter of each hole of the wire mesh used is 5cm x 5cm. The mice were placed under the wire mesh upside down, and the drop time of the mice was recorded. , which can be used as a bonus item in statistics.

(3) Climbing pole experiment

In order to evaluate the coordination ability of the limbs of the mice, the mice were placed on the spherical end (top) of a metal rod with a height of 60 cm, and the time for the mice to climb down from the top to the ground was recorded.

(4) Open field experiment

The open field experiment is a commonly used behavioral experiment in mice, which can detect the spontaneous activity and exploratory behavior of mice. To generate curiosity to explore new places; in order to evaluate the autonomous movement and avoidance of experimental animals in the new environment, the animals were placed in the center of the bottom surface of the box, and video and timing were performed at the same time. After observing for 5 minutes, stop the camera, clean the inner wall and bottom of the square box, so as not to affect the results of the next test due to the information left by the last animal, change the animal, continue the experiment, record the distance of the mice moving along the edge and the time spent exploring the middle part for statistics.

Claims (10)

1. The application of a monoclonal antibody in the preparation of a medicine for treating Parkinson's disease, the monoclonal antibody is an anti-serine 129 phosphorylated alpha synuclein (p-α-synuclein, p-α-syn) monoclonal antibody (C140S). 2. The application according to claim 1, wherein the monoclonal antibody is of type: IgG, and the antibody subtype is: IgG2b. 3. The use according to claim 1, wherein the medicament is in any ingestible formulation. 4. The use according to claim 1, wherein the formulation is in the form of parenteral administration. 5. The use according to claim 1, wherein the formulation is in the form of an injection. 6. The use according to claim 1, wherein the use is that the antibody can block the transmission of p-α-syn from cell to cell. 7. The use according to claim 1, wherein the use is that the antibody can pass through the blood-brain barrier and enter the brain parenchyma within 6 hours. 8. The application according to claim 1, wherein the application is that the antibody can improve the inversion of the ratio of CD4+/CD8+ T cells, alleviate the behavioral disorder of the transgenic mice, and increase the rotarod residence time of the transgenic mice , increased the limb strength of mice, and enhanced the coordination ability of transgenic mice. 9. The use according to claim 1, wherein the use is that the antibody can neutralize a toxic protein and reduce its cytotoxicity. 10. The application according to claim 1, wherein the application is that the antibody can reduce the p-α-syn content in the brain of the transgenic animal, increase the TH content in the brain, and alleviate the avoidance barrier of the transgenic mouse, illustrating This antibody C140S has neuroprotective effect and has therapeutic effect on PD model.

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